Multiple-mouse magnetic resonance imaging with cryogenic radiofrequency probes for evaluation of brain development.

Multiple-mouse magnetic resonance imaging (MRI) increases scan throughput by imaging several mice simultaneously in the same magnet bore, enabling multiple images to be obtained in the same time as a single scan. This increase in throughput enables larger studies than otherwise feasible and is particularly advantageous in longitudinal study designs where frequent imaging time points result in high demand for MRI resources. Cryogenically-cooled radiofrequency probes (CryoProbes) have been demonstrated to have significant signal-to-noise ratio benefits over comparable room temperature coils for in vivo mouse imaging. In this work, we demonstrate implementation of a multiple-mouse MRI system using CryoProbes, achieved by mounting four such coils in a 30-cm, 7-Tesla magnet bore. The approach is demonstrated for longitudinal quantification of brain structure from infancy to early adulthood in a mouse model of Sanfilippo syndrome (mucopolysaccharidosis type III), generated by knockout of the Hgsnat gene. We find that Hgsnat-/- mice have regionally increased growth rates compared to Hgsnat+/+ mice in a number of brain regions, notably including the ventricles, amygdala and superior colliculus. A strong sex dependence was also noted, with the lateral ventricle volume growing at an accelerated rate in males, but several structures in the brain parenchyma growing faster in females. This approach is broadly applicable to other mouse models of human disease and the increased throughput may be particularly beneficial in studying mouse models of neurodevelopmental disorders.

Brain mapping across 16 autism mouse models reveals a spectrum of functional connectivity subtypes.

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.

Neuroanatomy and behavior in mice with a haploinsufficiency of AT-rich interactive domain 1B (ARID1B) throughout development.

BACKGROUND: One of the causal mechanisms underlying neurodevelopmental disorders (NDDs) is chromatin modification and the genes that regulate chromatin. AT-rich interactive domain 1B (ARID1B), a chromatin modifier, has been linked to autism spectrum disorder and to affect rare and inherited genetic variation in a broad set of NDDs. METHODS: A novel preclinical mouse model of Arid1b deficiency was created and validated to characterize and define neuroanatomical, behavioral and transcriptional phenotypes. Neuroanatomy was assessed ex vivo in adult animals and in vivo longitudinally from birth to adulthood. Behavioral testing was also performed throughout development and tested all aspects of motor, learning, sociability, repetitive behaviors, seizure susceptibility, and general milestones delays. RESULTS: We validated decreased Arid1b mRNA and protein in Arid1b+/- mice, with signatures of increased axonal and synaptic gene expression, decreased transcriptional regulator and RNA processing expression in adult Arid1b+/- cerebellum. During neonatal development, Arid1b+/- mice exhibited robust impairments in ultrasonic vocalizations (USVs) and metrics of developmental growth. In addition, a striking sex effect was observed neuroanatomically throughout development. Behaviorally, as adults, Arid1b+/- mice showed low motor skills in open field exploration and normal three-chambered approach. Arid1b+/- mice had learning and memory deficits in novel object recognition but not in visual discrimination and reversal touchscreen tasks. Social interactions in the male-female social dyad with USVs revealed social deficits on some but not all parameters. No repetitive behaviors were observed. Brains of adult Arid1b+/- mice had a smaller cerebellum and a larger hippocampus and corpus callosum. The corpus callosum increase seen here contrasts previous reports which highlight losses in corpus callosum volume in mice and humans. LIMITATIONS: The behavior and neuroimaging analyses were done on separate cohorts of mice, which did not allow a direct correlation between the imaging and behavioral findings, and the transcriptomic analysis was exploratory, with no validation of altered expression beyond Arid1b. CONCLUSIONS: This study represents a full validation and investigation of a novel model of Arid1b+/- haploinsufficiency throughout development and highlights the importance of examining both sexes throughout development in NDDs.

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

Prenatal ß-catenin/Brn2/Tbr2 transcriptional cascade regulates adult social and stereotypic behaviors.

Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a ß-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the ß-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

Regional brain volumes changes in adult male FMR1-KO mouse on the FVB strain.

Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disorder (ASD). FMR1-KO mice mimic the etiology and phenotypic manifestations of FXS. Neuroanatomical changes in specific brain regions have been reported in clinical settings and in preclinical models. FMR1-KO mice have been generated in different strains including C57Bl/6 (B6) and FVB. Mice on different genetic backgrounds have stable yet distinct behavioral phenotypes that may lead to unique gene-strain interactions on brain structure. Previous magnetic resonance imaging (MRI) studies have examined FMR1 knockout male mice on a B6 and found few differences compared to wild-type mice. Here, we examine brain volumes in FMR1 knockout male mice on a FVB background using high resolution (multi-channel 7.0Tesla) MRI. We observe multiple differences in the neuroanatomy of male FMR1-/y mice on a FVB background. Significantly larger relative volume (% total brain volume) differences were found in major white matter structures throughout the brain. In addition, there were changes in areas associated with fronto-striatal circuitry and other regions. Functional and structural connectivity differences are often seen in human ASD, and therefore, this increased white matter seen in the FMR1-KO-FVB could be highlighting a structural over-connectivity, which could lead to some of the behavioral abnormalities seen with the FMR1-KO-FVB mice. Furthermore, these results highlight the importance of genetic strain contribution to brain structure.

Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity.

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1-2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Altered brain development in an early-onset murine model of Alzheimer's disease.

Murine models of Alzheimer's disease (AD) have been used to draw associations between atrophy of neural tissue and underlying pathology. In this study, the early-onset TgCRND8 mouse model of AD and littermate controls were scanned longitudinally with in vivo manganese-enhanced MRI (MEMRI) before and after the onset of amyloid plaque deposition at 12 weeks of age. Separate cohorts of mice were scanned at 1 week (ex vivo imaging) and 4 weeks (MEMRI) of age to investigate early life alterations in the brain. Contrary to our expectations, differences in neuroanatomy were found in early post-natal life, preceding plaque deposition by as much as 11 weeks. Many of these differences remained at all imaging time points, suggesting that they were programmed early in life and were unaffected by the onset of pathology. Furthermore, rather than showing atrophy, many regions of the TgCRND8 brain grew at a faster rate compared to controls. These regions contained the greatest density of amyloid plaques and reactive astrocytes. Our findings suggest that pathological processes as well as an alteration in brain development influence the TgCRND8 neuroanatomy throughout the lifespan.

Neuroanatomical phenotypes in a mouse model of the 22q11.2 microdeletion.

Recurrent deletions at the 22q11.2 locus have been established as a strong genetic risk factor for the development of schizophrenia and cognitive dysfunction. Individuals with 22q11.2 deletions have a range of well-defined volumetric abnormalities in a number of critical brain structures. A mouse model of the 22q11.2 deletion (Df(16)A(+/-)) has previously been utilized to characterize disease-associated abnormalities on synaptic, cellular, neurocircuitry, and behavioral levels. We performed a high-resolution MRI analysis of mutant mice compared with wild-type littermates. Our analysis revealed a striking similarity in the specific volumetric changes of Df(16)A(+/-) mice compared with human 22q11.2 deletion carriers, including in cortico-cerebellar, cortico-striatal and cortico-limbic circuits. In addition, higher resolution magnetic resonance imaging compared with neuroimaging in human subjects allowed the detection of previously unknown subtle local differences. The cerebellar findings in Df(16)A(+/-) mice are particularly instructive as they are localized to specific areas within both the deep cerebellar nuclei and the cerebellar cortex. Our study indicates that the Df(16)A(+/-)mouse model recapitulates most of the hallmark neuroanatomical changes observed in 22q11.2 deletion carriers. Our findings will help guide the design and interpretation of additional complementary studies and thereby advance our understanding of the abnormal brain development underlying the emergence of 22q11.2 deletion-associated psychiatric and cognitive symptoms.

Macroorchidism and testicular fibrosis associated with autoimmune thyroiditis.

A 16-year-old male with long-standing atrophic chronic lymphocytic thyroiditis was evaluated for macroorchidism. A testicular biopsy prior to treatment revealed peritubular and interstitial fibrosis, reduced spermatogenesis and sparse Leydig cells with nonprominent smooth endoplasmic reticulum. Biological/immunological LH and FSH ratios were reduced, I-LH and FSH response to GnRH was blunted, and levels of testosterone and androstenedione were low. Twenty-two months after thyroid treatment, the testicular size was unchanged, and the degree of fibrosis showed minimal regression. Spermatogenesis with normal morphology was present, Leydig cells with Reinke crystals were present, and surface area and diameter of the seminiferous tubules had increased only slightly. There was a normal I-LH and FSH response to GnRH, and normal levels of testosterone and androstenedione. This study, along with previous reports, suggests that the etiology of the hypothyroid state may influence the development of testicular fibrosis.

Plasminogen in peritoneal fluid: a possible nonsteroidal indicator of ovulation.

Plasminogen levels in peritoneal fluid were evaluated in 40 fertile women with regular ovulatory cycles. Fifteen samples were obtained before ovulation and 25 samples after ovulation. Plasminogen content and concentration in postovulatory peritoneal fluid was 0.84 +/- 0.10 mg and 6.1 +/- 0.57 mg/dl, respectively. These values were significantly higher than in the preovulatory peritoneal fluid, which were 0.36 +/- 0.12 mg (P less than 0.005) and 3.5 +/- 0.33 mg/dl, (P less than 0.001), respectively. Estradiol and progesterone (P) concentrations in peritoneal fluid were significantly higher after ovulation (P less than 0.05 and P less than 0.01, respectively). P concentration in serum and peritoneal fluid showed a linear correlation (P less than 0.05). Peritoneal fluid plasminogen is significantly elevated after ovulation and may be a nonsteroidal marker for follicular rupture and oocyte extrusion.

The effects of single-dose luteinizing hormone-releasing hormone on ovulatory menstrual function: support for a single luteinizing hormone- and follicle-stimulating hormone-releasing factor.

Ten ovulatory women were followed with gonadotropin and steroid determinations through two cycles. They were given 500 micrograms of luteinizing hormone-releasing hormone (LH-RH) during the periovulatory period of the second cycle to determine whether ovulation could be facilitated without altering corpus luteum function. Successive cycles demonstrated concordance for patterns of gonadotropin and steroid secretion when studied as group means. Two control cycles and one treatment cycle were consistent with luteal phase defects. The use of a supramaximal dose of LH-RH in these women neither facilitated ovulation nor adversely affected luteal function. A significant linear correlation was noted between peak LH and follicle-stimulating hormone (FSH) values for the spontaneous surges. This same linear relationship was maintained for the LH and FSH responses to 500 micrograms LH-RH. The present data produce further evidence demonstrating that the secretion of LH and FSH appears to be modulated by gonadal steroids and under the permissive control of a single gonadotropin-releasing hormone.

Serum progesterone, 17 alpha-hydroxyprogesterone, human chorionic gonadotropin, and prolactin in early pregnancy and a case of spontaneous abortion.

Sequential serum samples were obtained from each of seven healthy volunteers whose dates of conception were carefully monitored, with the use of artificial insemination techniques. Progesterone, 17 alpha-hydroxyprogesterone, beta-hCG, and prolactin were measured by radioimmunoassay, utilizing specific antibodies. Of the seven initial volunteers, six women delivered viable, healthy infants. The seventh woman aborted spontaneously during the 10th week after conception. In the control patients, the levels for beta-hCG rose in a predictable fashion, rising to mean peak levels (10,000 mIU/ml) by the 8th week after conception. Progesterone concentrations gradually increased and plateaued from the 3rd to the 9th week. Mean progesterone values during that time period ranged from 20.6 to 24.6 ng/ml. The mean peak values of 4 ng/ml 17 alpha-hydroxyprogesterone was reached by the 3rd week after conception, gradually declining to luteal levels by the 8th week after conception. Prolactin remained below 30 ng/ml until the 7th week after conception, increasing gradually thereafter. This study failed to show that 17 alpha-hydroxyprogesterone was a reliable marker of corpus luteum function in pregnancy. Endocrine parameters reliably predicted fetal jeopardy in the abortion patient.

Maternal and amniotic fluid steroids throughout human pregnancy.

Concentrations of testosterone, dihydrotestosterone, androstenedione, progesterone, 17 alpha-hydroxyprogesterone, and estradiol were measured by radioimmunoassay in the amniotic fluid and maternal peripheral blood obtained from normal pregnancies between 14 and 40 weeks of gestation. There was a sex difference in the levels of all the androgenic steroids in the amniotic fluid before 20 weeks with higher levels in pregnancies with male fetuses. Amniotic fluid 17 alpha-hydroxyprogesterone levels were significantly elevated in a pregnancy with the fetus affected with congenital adrenal hyperplasia. The levels of all the steroids in the amniotic fluid were significantly elevated in the pregnancy with molar degeneration of the placenta. There was a sex difference in the levels of dihydrotestosterone in the maternal peripheral blood before 20 weeks with higher levels in pregnancies with male fetuses. There was no correlation between the steroid levels in the maternal serum and amniotic fluid even though most of the samples of maternal serum were drawn at the same time as amniocentesis.

Serum levels of estrone and estradiol after implantation of estradiol pellets.

Serum estrone, estradiol, progesterone, FSH, and LH levels were studied after the implantation of estradiol pellets in female volunteers in the reproductive age group. The estradiol pellets were implanted at six-month intervals. The first implant consisted of four pellets (25 mg each). Subsequent implants consisted of a reduction by one pellet as compared to the previous implant. The mean serum estradiol levels during the six-month period of implantation or at the end of six-month intervals were in the premenopausal range and did not differ significantly during the study. The serum estrone levels showed a tendency to increase with time even though the number of pellets implanted was decreasing, apparently a result of incomplete absorption of the implanted estradiol during the six-month period. The results of serum progesterone determinations and basal body temperature records indicate that the suppression of ovulation takes place most consistently during the second and subsequent months after estradiol pellet implantation.

Accumulation of norethindrone and individual metabolites in human plasma during short- and long-term administration of a contraceptive dosage.

Blood levels of free, sulfate, and glucuronide conjugates of norethindrone (NE) and its ring A reduced metabolites 17alpha-ethynyl-5beta-estrane-3alpha, 17beta-diol and 17alpha-ethynyl-5alpha-estrane-3alpha, 17beta-diol were measured in a female volunteer who received six consecutive daily doses of 2.5 mg. of NE and in four female volunteers undergoing chronic treatment with Orthonovum 2 mg. (2 mg. of NE and 0.1 mg. of mestranol [ME]). The blood levels were quantified by gas chromatograph-mass spectrometry. During treatment for 6 days with 2.5 mg. of NE daily, the 3 hour blood levels of NE and the ring A reduced metabolites increased in a stepwise fashion. During long-term treatment the concentrations of NE, NE sulfate, and the conjugates of the ring A reduced metabolites were seen to build up to a peak at approximately the midpoint of the treatment phase of each cycle, and drop to near baseline during the time when no drug was administered. Individuals varied as to their tendency to accumulate the drug and metabolites, and as to the relative proportion of metabolites formed.

PIP: The accumulation of norethindrone (NE) and its ring A reduced metabolites in human plasma during short- and long-term administration of the contraceptive Orthonovum (2 mg NE plus .1 mg mestranol) was investigated. Free, sulfate, and glucuronide conjugates of NA and 17 alpha-ethynyl-5 beta-estrane-3 alpha,17 beta-diol and 17 alpha-ethynyl-5 alpha-estrane-3 alpha,17 beta-diol were measured in a female volunteer who received 6 consecutive doses of 2.5 mg NE/day and in 4 female volunteers, aged 20-37 years, undergoing chronic treatment with Orthonovum. Gas chromatograph-mass spectrometry were used to measure the blood levels. During treatment for 6 days with 2.5 mg/day, the 3 hour blood levels of NE and the metabolites increased in a stepwise fashion. During long-term treatment, NE, NE sulfate, and the conjugates of the metabolites were built up to a peak about midpoint of the treatment phase of each cycle, and dropped to near baseline when no drug was administered. The individuals varied as to their tendency to accumulate the drug and metabolites, and the relative proportion of metabolites formed.